Abstract

By using chromosome conformation capture technology, a recent study has revealed two
alternative three-dimensional folding states of the human genome during the cell cycle.

The packaging conundrum at ever-higher levels of scrutiny

Understanding the spatial organization of chromosomes represents a major quest, not
only for our comprehension of how a length of 2 m of DNA can be packaged into a nucleus
of just a few microns, but also because the physical interactions occurring within
and between chromosomes are thought to play an important role in gene regulation,
DNA replication and genome stability. Over recent years, the development of chromosome
conformation capture (3C)-based techniques, together with the emergence of next-generation
sequencing, have changed our view of nuclear organization. High-throughput conformation
capture techniques have been developed to study the physical interactions of the chromatin
fiber with or within genomic regions spanning from a few megabases (5C technique)
to a whole-genome scale (4C and Hi-C techniques). In 2009, Eric Lander, Job Dekker
and colleagues [1] described the first human chromosomal architecture at a resolution of 1 Mb. Then,
four years later, owing to the rapid evolution of sequencing capabilities, Bing Ren
and colleagues [2] published a high-resolution map of the ‘chromatin interactome’ in human fibroblasts
at a resolution of 5 to 10 kb.

The picture that emerges from this blossoming area of research is that metazoan chromosomes
are characterized by a nested hierarchy of structural layers (Figure 1, left panel). Within each chromosome territory, compartments originally termed ‘A’
and ‘B’, each of several megabases, tend to associate within each single chromosome,
reflecting the preferential colocalization of active, gene-rich regions and their
segregation from gene-poor, inactive regions [1]. More recently, a further sub-megabase scale of genome partitioning into topologically
associating domains (TADs) has been reported [3-5]. Sequences within TADs tend to interact more frequently than with any other surrounding
region of the genome. These domains, spanning a few hundred kilobases, are stable
across different cell types, suggesting that they are an inherent property of mammalian
genomes. Furthermore, within TADs, a network of cell-type-specific physical interactions
between potential regulatory sequences has been shown to take place [6]. Consistent with this, a recent study using high-resolution Hi-C showed that enhancer-promoter
contacts almost always occur within TADs [2].

Figure 1.Cell-cycle dependence of chromosome structure. Left: high-resolution chromosome-conformation capture technologies have revealed
that each mammalian chromosome territory can be decomposed into multi-megabase compartments
where preferential interactions occur between active gene-rich and inactive gene-poor
regions of the chromosome. Compartments can be further partitioned at the submegabase
scale into topological associating domains (TADs) spanning a few hundred kilobases
each. Dekker and colleagues [7] show that compartments and TADs are stable across the cell cycle from G1 to late
S phase. Right: in metaphase, the compartment and TAD structures disappear and are
replaced by a cylindrically condensed structure of linearly arranged short-range loops
[7], which occur on a shorter genomic scale (80 kb on average) than TADs. It is hypothesized
that the persistence of bookmarking factors on the mitotic chromosome might facilitate
the rapid re-emergence of compartments and TADs upon exit of the cells from mitosis.

Despite the pace at which new details of mammalian chromosome organization are accumulating,
many fundamental questions remain unanswered. For example, what is the cell-to-cell
variability in the structures that give rise to compartments, TADs and promoter-enhancer
interactions? How important is each of those structural layers for the regulation
of transcription? What are the molecular mechanisms that determine the appearance
of TADs and compartments? When are these layers established during development? Are
these different levels of chromosome organization present throughout the cell cycle,
or are they established at a particular phase? Finally, what is the structure of mitotic
chromosomes? Are the hierarchical layers of folding maintained during mitosis, or
do chromosomes acquire a different organization, as suggested by light and electron
microscopy? A recent exciting study has shed light on these last questions [7].

Compartments and TADs are erased during mitosis

Although DNA fluorescence in situ hybridization (FISH) coupled to super-resolution microscopy has previously been used
in murine cells to investigate the presence of TADs within the X-inactivation center
during the cell cycle from G1/S phase to mitotic prophase [4], a detailed molecular analysis of chromatin interactions throughout the cell cycle
had not, until recently, been reported. Dekker and colleagues [7] now report the results of performing carbon-copy chromosome conformation capture
(5C) and Hi-C experiments in synchronized human HeLa cells at different phases of
the cell cycle. Using 5C technology spanning the whole of chromosome 21 at a resolution
of 250 kb, the authors first studied the long-range chromatin interactions of early
G1-, mid-G1-, S- and M-phase cells. Interaction maps of these different stages showed
that the mitotic interaction pattern differs dramatically from that at all other stages,
thus revealing two distinct chromosome folding states during the cell cycle. Using
Hi-C (at a resolution of 40 kb) on mitotic and mid-G1-stage cells [7], the investigators further found that both compartments [1] and TADs [3,4] appear to be absent throughout the entire genome in metaphase, becoming detectable
only in early G1 phase and remaining unchanged throughout interphase. These observations
on mitotic chromosomes were repeated and validated in another cell line and in primary
human fibroblasts. Importantly, the authors showed that, apart from metaphase, compartments
and TADs are not restricted to any specific cell cycle phase and they are easily detected
in early G1, thus ruling out the possibility that the patterns observed in 5C or Hi-C
could be due to a superposition of alternative cell-cycle phase-specific conformations.

The finding that TADs and compartments apparently disappear during metaphase, and
reappear in early G1, raises the questions of why they are lost and also what mechanism
ensures their prompt re-establishment upon exit from mitosis. It is known that certain
transcription factors and histone modifications or associated factors can act as ‘bookmarking’
factors on mitotic chromosomes [8] by remaining bound to the condensed chromatin polymer in order to ensure the propagation
of transcriptional states to daughter cells. It is therefore tempting to speculate
that structural bookmarking factors that propagate organizational information to daughter
cells might similarly exist. Although the molecular bases of compartments and TADs
remain elusive, accumulating evidence suggests that long-range interactions between
genomic sites bound by the proteins CTCF and cohesin contribute to the organization
of chromatin architecture at the TAD scale, either by associating with TAD boundaries
[3] or by supervising a network of long-range interactions inside single TADs that might
stabilize the structure within TADs [6]. Interestingly, both CTCF and cohesin have been shown to associate with mitotic chromosomes
[8], suggesting that mutual interactions between CTCF and/or cohesin binding sites might
readily occur upon exit from mitosis, thus enabling TADs to be established immediately
at the beginning of G1 phase. Other factors that might remain bound during mitosis
to the positions of TAD or compartment boundaries include Polycomb group proteins,
which have been shown to significantly overlap with TAD boundaries [5] on mitotic chromosomes in Drosophila[9]

A polymer view of a mitotic chromosome

The apparent loss of partitioning into TADs and compartments observed in mitotic chromosomes
by Dekker and colleagues [7] suggests that they contain unusually few structural details compared with those of
their interphase counterparts. However, the authors cleverly exploited the quantitative
information present in the Hi-C mitotic chromosome data to build a structural model.
The foundation of their approach is that Hi-C or 5C interaction maps represent the
frequency at which every pair of genomic loci along the chromatin polymer encounter
each other, averaged over millions of cells. It is therefore possible to use models
from polymer physics to simulate virtual Hi-C (or 5C) data and compare them with data
from experiments. Using such an approach, the thermodynamic ensemble of configurations
of a model chromosome can be generated by computer simulations, and a contact map
is retrieved by averaging the contacts of each pair of loci over all simulated polymer
conformations, allowing a straightforward comparison with experimental data.

In the case of mitotic chromosome data, the Hi-C or 5C contact maps are homogeneous,
with no sign of the regular patterning due to TADs and compartments that characterize
the interphase contact maps [7]. Dekker and colleagues also noted that the contact probability between two loci gradually
decreases with increasing genomic distance (albeit significantly more slowly than
on interphase chromosomes), and then suddenly falls off to zero at approximately 10 Mb.
This unusual behavior cannot be explained by any simple polymer models. Building on
the well-established experimental evidence describing the quantitative properties
of mitotic chromosomes (such as cylindrical symmetry, linear organization of chromatids
and chromatin packing density), the authors thus set out to build various alternative
models for the chromatin organization within mitotic chromosomes, simulated all of
them and tested each model’s predictions. In order to reproduce the unexpected fall-off
in contact probabilities for loci that are separated by >10 Mb, they had to impose
that the chromatin fiber is linearly organized, so that genomic loci belonging to
distal parts of the chromosome (that is, separated by >10 Mb) cannot be brought into
close spatial proximity. Furthermore, they had to impose that the chromatin fiber
is arranged in an array of consecutive loops, each spanning approximately 80 kb, in
order for their simulations to account correctly for the gentle decrease in contact
frequencies in the genomic length range between 0 and 10 Mb. Thus, amongst all possible
models tested, it was this combination of linear organization and consecutive looping
that best accounted for the behavior of the contact probability of mitotic chromosomes
over all genomic length scales. Interestingly, the authors further discovered that
they could simultaneously reproduce the scaling of contact probabilities and the homogeneity
of the mitotic contact maps only when they allowed the size of the loops to be different
(and stochastic) in each of the polymer configurations.

Future perspectives

The study by Dekker and colleagues represents a powerful combination of biochemical
investigation and polymer modeling [7]. The prowess of such an approach is that it can provide insights into structure that
would not have been obtained without modeling. The picture that emerges, thanks to
their study, is that of a mitotic chromosome composed of consecutive loops of chromatin,
the size of which is on average 80 kb, although this varies from cell to cell, which
are arranged in a linear fashion eventually resulting in an effective cylindrical
chromosomal volume (Figure 1, right panel). This picture is both qualitatively and quantitatively in agreement
with previously proposed ‘loops-on-a-scaffold’ models of mitotic chromosomes based
on microscopy and biochemical assays.

Although the molecular mechanisms that could mediate this pervasive local looping
remain elusive, the model predicts that cell-to-cell variability of looping events
has a sizeable effect in organizing the structure of mitotic chromatin. Indeed, the
potential importance of fluctuations in interphase chromosome structure was also recently
revealed by an adaptation of the Hi-C technique to single cells [10]. In the future, this type of single-cell approach, together with physical modeling
and quantitative analysis, could allow the exploration of chromosome structure in
situations where cell-to-cell variability might be crucial, such as during early development.